Epigenetic inheritance plays an important but poorly understood role in regulating cell development and differentiation. Efforts to elucidate epigenetic mechanisms have identified the post-translational modification of histone proteins as a major transducer of epigenetic information. The effect these modifications have on epigenetic control occurs either through biophysical alteration of the chromatin fiber or through the ability to recruit effector proteins to the site of modification. In recent years the application of sensitive and high throughput mass spectrometry (MS)-based proteomic techniques to characterize histone modifications has revealed a large number of novel modifications or sites of modification on these proteins. However, these studies used histones that were purified from asynchronously growing yeast or mammalian cells. As a consequence, important modifications that normally exist at low levels during specific phases of the cell cycle could easily have been missed since they would not be significantly enriched in the total pool of histones purified. Furthermore, highly labile modifications such as ubiquitylation and sumoylation are highly likely to have been missed in these early studies since standard histone extraction and purification protocols do not prevent enzymatic removal of these labile modifications. To circumvent these technical issues, we have created a series of budding yeast strains that have one copy of each histone gene genomically tagged with an epitope sequence that allows denaturing protein purification. We will use these strains to purify under denaturing conditions the individual histones, and the two histone variants Htz1 and Cse4, during each phase of the cell cycle. To detect the maximum coverage of possible PTM sites at each individual histone, these purified histones will be analyzed by using both "top-down" and "bottom-up" MS-based approaches in a complimentary way to characterize the full complement of modifications residing on these histones at discreet cell-cycle phases. These approaches will allow us to identify novel modifications and/or sites of modification that normally would be undetectable due to either their labile nature or insufficient abundance. This proposal thus aims to create a eukaryotic "epigenomic atlas" that will define the full complement of histone modifications that exist on yeast histones throughout the cell cycle. Our study will not only facilitate the identification of novel marks that can be further analyzed in human stem cells under the epigenomics initiative, but it also will provide valuable information into the dynamics of histone modifications that influence chromosome biology during cell division. This work will therefore serve as a foundation for understanding the complex epigenetic regulation that occurs in humans, and how dysfunctional regulation promotes pathological processes such as cancer. PUBLIC HEALTH RELEVANCE: Defects in chromatin organization, DNA packaging and its accessibility is a major cause of human disease, including cancer. These proposed studies aim to identify novel epigenetic histone marks, which will undoubtedly turn out to be central regulators of DNA-based activities. This knowledge will help to address the underlying causes of cancer and other public health concerns.